Effects of proline mutations on the folding of staphylococcal nuclease

Effects of proline isomerizations on the equilibrium unfolding and kinetic refolding of staphylococcal nuclease were studied by circular dichroism in the peptide region (225 nm) and fluorescence spectra of a tryptophan residue. For this purpose, four single mutants (P11A, P31A, P42A, and P56A) and four multiple mutants (P11A/P47T/P117G, P11A/P31A/P47T/P117G, P11A/P31A/P42A/P47T/P117G, and P11A/P31A/P42A/P47T/P56A/P117G) were constructed. These mutants, together with the single and double mutants for Pro47 and Pro117 constructed in our previous study, cover all six proline sites of the nuclease. The P11A, P31A, and P42A mutations did not change the stability of the protein remarkably, while the P56A mutation increased protein stability to a small extent by 0.5 kcal/mol. The refolding kinetics of the protein were, however, affected remarkably by three of the mutations, namely, P11A, P31A, and P56A. Most notably, the amplitude of the slow phase of the triphasic refolding kinetics of the nuclease observed by stopped-flow circular dichroism decreased by increasing the number of the proline mutations; the slow phase disappeared completely in the proline-free mutant (P11A/P31A/P42A/P47T/P56A/P117G). The kinetic refolding reactions of the wild-type protein assessed in the presence of Escherichia coli cyclophilin A showed that the slow phase was accelerated by cyclophilin, indicating that the slow phase was rate-limited by cis-trans isomerization of the proline residues. Although the fast and middle phases of the refolding kinetics were not affected by cyclophilin, the amplitude of the middle phase decreased when the number of the proline mutations increased; the percent amplitudes for the wild-type protein and the proline-free mutants were 43 and 13%, respectively. In addition to these three phases detected with stopped-flow circular dichroism, a very fast phase of refolding was observed with stopped-flow fluorescence, which had a shorter dead time (3.6 ms) than the stopped-flow circular dichroism. The following conclusions were drawn. (1) The effects of the P11A, P31A, and P56A mutations on the refolding kinetics indicate that the isomerizations of the three proline residues are rate-limiting, suggesting that the structures around these residues (Pro11, Pro31, and Pro56) may be organized at an early stage of refolding. (2) The fast phase corresponds to the refolding of the native proline isomer, and the middle phase whose amplitude has decreased when the number of proline mutations was increased may correspond to the slow refolding of non-native proline isomers. The occurrence of the fast- and slow-refolding reactions together with the slow phase rate-limited by the proline isomerization suggests that there are parallel folding pathways for the native and non-native proline isomers. (3) The middle phase did not completely disappear in the proline-free mutant. This suggests that the slow-folding isomer is produced not only by the proline isomerizations but also by another conformational event that is not related to the prolines. (4) The very fast phase detected with the fluorescent measurements suggests that there is an intermediate at a very early stage of kinetic refolding.     

The Pro117 to glycine mutation of staphylococcal nuclease simplifies the unfolding-folding kinetics

Kinetics of unfolding and refolding of a staphylococcal nuclease mutant, in which Pro117 is replaced by glycine, have been investigated by stopped-flow circular dichroism, and the results are compared with those for the wild-type protein. In contrast to the biphasic unfolding of the wild-type nuclease, the unfolding of the mutant is represented by a single-phase reaction, indicating that the biphasic unfolding for the wild-type protein is caused by cis-trans isomerization about the prolyl peptide bond in the native state. The proline mutation also simplifies the kinetic refolding. Importance of the results in elucidating the folding mechanism is discussed.     

Identification of proline residues responsible for the slow folding kinetics in pectate lyase C by mutagenesis

The folding mechanism of pectate lyase C (pelC) involves two slow phases that have been attributed to proline isomerization. To have a more detailed and complete understanding of the folding mechanism, experiments have been carried out to identify the prolyl-peptide bonds responsible for the slow kinetics. Site-directed mutagenesis has been used to mutate each of the prolines in pelC to alanine or valine. It has been determined that isomerization of the Leu219-Pro220 peptide bond is responsible for the slowest folding phase observed. The mutant P220A shows kinetic behavior that is identical to the wild-type protein except that the 46-s phase is eliminated. The Leu219-Pro220 peptide bond is cis in the native enzyme. An analysis of the free energy of unfolding of this mutant indicates that the mutation destabilizes the protein by about 4 kcal/mol. However, it appears that the major refolding pathways are unaltered. Further mutations were carried out in order to assign the peptide bond responsible for the 21-s folding phase in pelC. Mutation of the remaining 11 prolines, which are trans in the native enzyme, resulted in no significant changes in the kinetic folding behavior. The conclusion from these experiments is that the 21-s phase involves isomerization of more than one prolyl-peptide bond with similar activation energies.     

Effects of proline mutations on the unfolding and refolding of human lysozyme: the slow refolding kinetic phase does not result from proline cis-trans isomerization

The unfolding and refolding kinetics of six proline mutants of the human lysozyme (h-lysozyme) were carried out and compared to that of the wild-type protein. Our results show that the slow refolding phase observed in the h-lysozyme refolding kinetics cannot be ascribed to proline isomerization reactions. The h-lysozyme contains two proline residues at positions 71 and 103, both in the trans conformation in the native state. The refolding kinetics of the P71G/P103G mutant, in which both prolines have been replaced by a glycine, were found to be similar to those of the wild-type protein. The same slow phase amplitude of about 10% was found for both proteins, and the slow phase rate constants were also identical within experimental error. Other mutants such as P103G or P71G, in which only one of the two prolines has been replaced by a glycine, and A47P with its three prolines, gave identical slow refolding phases. The X-ray structure analysis and scanning microcalorimetric study of each protein (Herning et al., unpublished experiments) have confirmed that none of the considered mutations affects significantly protein structure and that no major changes in protein stability were brought about by these mutations. Therefore, comparison of the properties of the mutant and wild-type proteins is legitimate. Interestingly, the refolding kinetics of the V110P mutant, in which a proline residue has been introduced at position 110 (N-terminus of an alpha-helix), were clearly triphasic. For this mutant an additional very slow phase with properties similar to those expected from the proline hypothesis was detected. Equilibrium denaturation studies were conducted for each protein, and the refolding pathway of h-lysozyme is partly presented. We also discuss the effect of proline mutations on the energetics of the folding pathway of the h-lysozyme in water.     

Replacement of a cis proline simplifies the mechanism of ribonuclease T1 folding

The refolding of ribonuclease T1 is dominated by two major slow kinetic phases that show properties of proline isomerization reactions. We report here that the molecular origin of one of these processes is the trans----cis isomerization of the Ser54-Pro55 peptide bond, which is cis in the native protein but predominantly trans in unfolded ribonuclease T1. This is shown by a comparison of the wild type and a designed mutant protein where Ser54 and Pro55 were replaced by Gly54 and Asn55, respectively. This mutation leaves the thermal stability of the protein almost unchanged; however, in the absence of Pro55 one of the two slow phases in folding is abolished and the kinetic mechanism of refolding is dramatically simplified.     

Multiple roles of prolyl residues in structure and folding

To explore the ways that proline residues may influence the conformational options of a polypeptide backbone, we have characterized Pro-->Ala mutants of cellular retinoic acid-binding protein I (CRABP I). While all three Xaa-Pro bonds are in the trans conformation in the native protein and the equilibrium stability of each mutant is similar to that of the parent protein, each has distinct effects on folding and unfolding kinetics. The mutation of Pro105 does not alter the kinetics of folding of CRABP I, which indicates that the flexible loop containing this residue is passive in the folding process. By contrast, replacement of Pro85 by Ala abolishes the observable slow phase of folding, revealing that correct configuration of the 84-85 peptide bond is prerequisite to productive folding. Substitution of Pro39 by Ala yields a protein that folds and unfolds more slowly. Removal of the conformational constraint imposed by the proline ring likely raises the transition state barrier by increasing the entropic cost of narrowing the conformational ensemble. Additionally, the Pro-->Ala mutation removes a helix-termination signal that is important for efficient folding to the native state.     

The Pro to glycine mutation of staphylococcal nuclease simplifies the unfolding—folding kinetics

Kinetics of unfolding and refolding of a staphylococcal nuclease mutant, in which Pro¹¹⁷ is replaced by glycine, have been investigated by stopped-flow circular dichroism, and the results are compared with those for the wild-type protein. In contrast to the biphasic unfolding of the wild-type nuclease, the unfolding of the mutant is represented by a single-phase reaction, indicating that the biphasic unfolding for the wild-type protein is caused by cis-trans isomerization about the prolyl peptide bond in the native state. The proline mutation also simplifies the kinetic refolding. Importance of the results in elucidating the folding mechanism is discussed.     

The rate-limiting step in the folding of the cis-Pro167Thr mutant of TEM-1 β-lactamase is the trans to cis isomerization of a non-proline peptide bond

The stability and kinetics of unfolding and refolding of the P167T mutant of the TEM-1 β-lactamase have been investigated as a function of guanidine hydrochloride concentration. The activity of the mutant enzyme was not significantly modified, which strongly suggests that the Glu166–Thr167 peptide bond, like the Glu166–Pro167, is cis. The mutation, however, led to a significant decrease in the stability of the native state relative to both the thermodynamically stable intermediate and the fully unfolded state of the protein. In contrast to the two slower phases seen in the refolding of the wild-type enzyme, only one phase was detected in the refolding of the mutant, indicating a determining role of proline 167 in the kinetics of folding of the wild-type enzyme. The former phases are replaced by rapid refolding when the enzyme is unfolded for short periods of time, but the latter is independent of the time of unfolding. The monophasic refolding reaction of the mutant is proposed to reflect mainly the trans→cis isomerization of the Glu166–Thr167 peptide bond. © 1996 John Wiley & Sons, Inc.     

Parallel channels and rate-limiting steps in complex protein folding reactions: prolyl isomerization and the alpha subunit of Trp synthase,a TIM barrel protein

A kinetic folding mechanism for the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, involving four parallel channels with multiple native, intermediate and unfolded forms, has recently been proposed. The hypothesis that cis/trans isomerization of several Xaa-Pro peptide bonds is the source of the multiple folding channels was tested by measuring the sensitivity of the three rate-limiting phases (tau(1), tau(2), tau(3)) to catalysis by cyclophilin, a peptidyl-prolyl isomerase. Although the absence of catalysis for the tau(1) (fast) phase leaves its assignment ambiguous, our previous mutational analysis demonstrated its connection to the unique cis peptide bond preceding proline 28. The acceleration of the tau(2) (medium) and tau(3) (slow) refolding phases by cyclophilin demonstrated that cis/trans prolyl isomerization is also the source of these phases. A collection of proline mutants, which covered all of the remaining 18 trans proline residues of alphaTS, was constructed to obtain specific assignments for these phases. Almost all of the mutant proteins retained the complex equilibrium and kinetic folding properties of wild-type alphaTS; only the P217A, P217G and P261A mutations caused significant changes in the equilibrium free energy surface. Both the P78A and P96A mutations selectively eliminated the tau(1) folding phase, while the P217M and P261A mutations eliminated the tau(2) and tau(3) folding phases, respectively. The redundant assignment of the tau(1) phase to Pro28, Pro78 and Pro96 may reflect their mutual interactions in non-random structure in the unfolded state. The non-native cis isomers for Pro217 and Pro261 may destabilize an autonomous C-terminal folding unit, thereby giving rise to kinetically distinct unfolded forms. The nature of the preceding amino acid, the solvent exposure, or the participation in specific elements of secondary structure in the native state, in general, are not determinative of the proline residues whose isomerization reactions can limit folding.     

Kinetic coupling of folding and prolyl isomerization of beta2-microglobulin studied by mutational analysis

β₂-Microglobulin (β2-m), a protein responsible for dialysis-related amyloidosis, adopts a typical immunoglobulin domain fold with the N-terminal peptide bond of Pro32 in a cis isomer. The refolding of β2-m is limited by the slow trans-to-cis isomerization of Pro32, implying that intermediates with a non-native trans-Pro32 isomer are precursors for the formation of amyloid fibrils. To obtain further insight into the Pro-limited folding of β2-m, we studied the Gdn-HCl-dependent unfolding/refolding kinetics using two mutants (W39 and P32V β2-ms) as well as the wild-type β2-m. W39 β2-m is a triple mutant in which both of the authentic Trp residues (Trp60 and Trp95) are replaced by Phe and a buried Trp common to other immunoglobulin domains is introduced at the position of Leu39 (i.e., L39W/W60F/W95F). W39 β2-m exhibits a dramatic quenching of fluorescence upon folding, enabling a detailed analysis of Pro-limited unfolding/refolding. On the other hand, P32V β2-m is a mutant in which Pro32 is replaced by Val, useful for probing the kinetic role of the trans-to-cis isomerization of Pro32. A comparative analysis of the unfolding/refolding kinetics of these mutants including three types of double-jump experiments revealed the prolyl isomerization to be coupled with the conformational transitions, leading to apparently unusual kinetics, particularly for the unfolding. We suggest that careful consideration of the kinetic coupling of unfolding/refolding and prolyl isomerization, which has tended to be neglected in recent studies, is essential for clarifying the mechanism of protein folding and, moreover, its biological significance.     

Cis proline mutants of ribonuclease A. II. Elimination of the slow-folding forms by mutation.

Ribonuclease A is known to form an equilibrium mixture of fast-folding (UF) and slow-folding (US) species. Rapid unfolding to UF is then followed by a reaction in the unfolded state, which produces a mixture of UF, USII, USI, and possibly also minor populations of other US species. The two cis proline residues, P93 and P114, are logical candidates for producing the major US species after unfolding, by slow cis <==> trans isomerization. Much work has been done in the past on testing this proposal, but the results have been controversial. Site-directed mutagenesis is used here. Four single mutants, P93A, P93S, P114A, and P114G, and also the double mutant P93A, P114G have been made and tested for the formation of US species after unfolding. The single mutants P114G and P114A still show slow isomerization reactions after unfolding that produce US species; thus, Pro 114 is not required for the formation of at least one of the major US species of ribonuclease A. Both the refolding kinetics and the isomerization kinetics after unfolding of the Pro 93 single mutants are unexpectedly complex, possibly because the substituted amino acid forms a cis peptide bond, which should undergo cis --> trans isomerization after unfolding. The kinetics of peptide bond isomerization are not understood at present and the Pro 93 single mutants cannot be used yet to investigate the role of Pro 93 in forming the US species of ribonuclease A. The double mutant P93A, P114G shows single exponential kinetics measured by CD, and it shows no evidence of isomerization after unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of multiple prolyl isomerization reactions on the stability and folding kinetics of the notch ankyrin domain: experiment and theory

Studies on the folding kinetics of the Notch ankyrin domain have demonstrated that the major refolding phase is slow, the minor refolding phase is limited by the isomerization of prolyl peptide bonds, and that unfolding is multiexponential. Here, we explore the relationship between prolyl isomerization and folding heterogeneity using a combination of experiment and simulation. Proline residues were replaced with alanine, both singly and in various combinations. These destabilizing substitutions combine to eliminate the minor refolding phase, although unfolding heterogeneity persists even when all seven proline residues are replaced. To test whether prolyl isomerization influences the major refolding phase, we modeled folding and prolyl isomerization as a system of sequential reactions. Simulations that use rate constants of the major folding phase of the Notch ankyrin domain to represent intrinsic folding indicate that even with seven prolyl isomerization reactions, only two significant phases should be observed, and that the fast observed phase provides a good approximation of the intrinsic folding in the absence of prolyl isomerization. These results indicate that the major refolding phase of the Notch ankyrin domain reflects an intrinsically slow folding transition, rather than coupling of fast folding events with slow prolyl isomerization steps. This is consistent with the observation that the single observed refolding phase of a construct in which all proline residues are replaced remains slow. Finally, the simulation fails to produce a second unfolding phase at high urea concentrations, indicating that prolyl isomerization does not play a role in the three-state mechanism that leads to this heterogeneity.     

Folding of intracellular retinol and retinoic acid binding proteins

The folding mechanisms of cellular retinol binding protein II (CRBP II), cellular retinoic acid binding protein I (CRABP I), and cellular retinoic acid binding protein II (CRABP II) were examined. These beta-sheet proteins have very similar structures and higher sequence homologies than most proteins in this diverse family. They have similar stabilities and show completely reversible folding at equilibrium with urea as a denaturant. The unfolding kinetics of these proteins were monitored during folding and unfolding by circular dichroism (CD) and fluorescence. During unfolding, CRABP II showed no intermediates, CRABP I had an intermediate with nativelike secondary structure, and CRBP II had an intermediate that lacked secondary structure. The refolding kinetics of these proteins were more similar. Each protein showed a burst-phase change in intensity by both CD and fluorescence, followed by a single observed phase by both CD and fluorescence and one or two additional refolding phases by fluorescence. The fluorescence spectral properties of the intermediate states were similar and suggested a gradual increase in the amount of native tertiary structure present for each step in a sequential path. However, the rates of folding differed by as much as 3 orders of magnitude and were slower than those expected from the contact order and topology of these proteins. As such, proteins with the same final structure may not follow the same route to the native state.     

Trigger factor-assisted folding of bovine carbonic anhydrase II

Spontaneous refolding of GdnHCl denatured bovine carbonic anhydrase II (BCA II) shows at least three phases: a burst phase, a fast phase, and a slow phase. The fast and slow phases are both controlled by proline isomerization. However, we find that in trigger factor (TF)-assisted BCA II folding, only the fast phase is catalyzed by wild-type TF, suggesting that certain proline residues are accessible in folding intermediates. The refolding yields of BCA II assisted by wild-type TF and TF mutants which lack PPIase activity are about the same, which provides further experimental evidence that the PPIase and chaperone activities of TF are independent. The binding of TF to folding intermediates during BCA II refolding was characterized by chemical crosslinking and Western blotting. A scheme for TF-assisted BCA II folding is proposed and the possible role of the TF dimer as a "binding" chaperone in vivo is discussed.     

Intramolecular catalysis of a proline isomerization reaction in the folding of dihydrofolate reductase.

The cis/trans isomerization of the peptide bond preceding proline residues in proteins can limit the rate at which a protein folds to its native conformation. Mutagenic analyses of dihydrofolate reductase (DHFR) from Escherichia coli show that this isomerization reaction can be intramolecularly catalyzed by a side chain from an amino acid which is distant in sequence but adjacent in the native conformation. The guanidinium NH2 nitrogen of Arg 44 forms one hydrogen bond to the imide nitrogen and a second to the carbonyl oxygen of Pro 66 in wild-type DHFR. Replacement of Arg 44 with Leu results in a change of the nature of the two slow steps in refolding from being limited by the acquisition of secondary and/or tertiary structure to being limited by isomerization. The simultaneous replacement of Pro 66 with Ala (i.e., the Leu 44/Ala 66 double mutant) eliminates this isomerization reaction and once again makes protein folding the limiting process. Apparently, one or both of the hydrogen bonds between Arg 44 and Pro 66 accelerate the isomerization of the Gln 65-Pro 66 peptide bond. The replacement of Arg 44 with Leu affects the kinetics of the slow folding reactions in a fashion which indicates that the crucial hydrogen bonds form in the transition states for the rate-limiting steps in folding.     

Cis-trans isomerization is rate-determining in the reactivation of denatured human carbonic anhydrase II as evidenced by proline isomerase.

The refolding of human carbonic anhydrase II is a sequential process. The slowest step involved is the recovery of enzymic activity (t1/2 = 9 min). Kinetic data from 'double-jump' measurements indicate that proline isomerization might be rate determining in the reactivation of the denatured enzyme. Proof of this is provided by the effect of proline isomerase on the reactivation kinetics: the presence of isomerase during reactivation lowers the half-time of the reaction to 4 min, and inhibition of proline isomerase completely abolishes this kinetic effect. A similar acceleration of the refolding process by proline isomerase is also observed for bovine carbonic anhydrase II, in contrast to what has previously been reported. In human carbonic anhydrase II there are two cis-peptidyl-Pro bonds at Pro30 and Pro202. Two asparagine single mutants (P30N and P202N) and a glycine double mutant (P30G/P202G) were constructed to investigate the role of these prolines in the rate limitation of the reactivation process. Both in the presence and absence of PPIase the P202N mutant behaved exactly like the unmutated enzyme. Thus, cis-trans isomerization of the Pro202 cis-peptidyl bond is not rate determining in the reactivation process. The mutations at position 30 led to such extensive destabilization of the protein that the refolding reaction could not be studied.     

Replacement of a conserved proline eliminates the absorbance-detected slow folding phase of iso-2-cytochrome c

As a test of the proline isomerization model, we have used oligonucleotide site-directed mutagenesis to construct a mutant form of iso-2-cytochrome c in which proline-76 is replaced by glycine [Wood, L. C., Muthukrishnan, K., White, T. B., Ramdas, L., & Nall, B. T. (1988) Biochemistry (preceding paper in this issue)]. For the oxidized form of Gly-76 iso-2, an estimate of stability by guanidine hydrochloride induced unfolding indicates that the mutation destabilizes the protein by 1.2 kcal/mol under standard conditions of neutral pH and 20 degrees C (delta G degrees u = 3.8 kcal/mol for normal Pro-76 iso-2 versus 2.6 kcal/mol for Gly-76 iso-2). The kinetics of folding/unfolding have been monitored by fluorescence changes throughout the transition region using stopped-flow mixing. The rates for fast and slow fluorescence-detected refolding are unchanged, while fast unfolding is increased in rate 3-fold in the mutant protein compared to normal iso-2. A new kinetic phase in the 1-s time range is observed in fluorescence-detected unfolding of the mutant protein. The presence of the new phase is correlated with the presence of species with an altered folded conformation in the initial conditions, suggesting assignment of the phase to unfolding of this species. The fluorescence-detected and absorbance-detected slow folding phases have been monitored as a function of final pH by manual mixing between pH 5.5 and 8 (0.3 M guanidine hydrochloride, 20 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding

The protein folding process is often in vitro rate‐limited by slow cis‐trans proline isomerization steps. Importantly, the rate of this process in vivo is accelerated by prolyl isomerases (PPIases). The archetypal PPIase is the human cyclophilin 18 (Cyp18 or CypA), and Arg 55 has been demonstrated to play a crucial role when studying short peptide substrates in the catalytic action of Cyp18 by stabilizing the transition state of isomerization. However, in this study we show that a R55A mutant of Cyp18 is as efficient as the wild type to accelerate the refolding reaction of human carbonic anhydrase II (HCA II). Thus, it is evident that the active‐site located Arg 55 is not required for catalysis of the rate‐limiting prolyl cis‐trans isomerization steps during the folding of a protein substrate as HCA II. Nevertheless, catalysis of cis‐trans proline isomerization in HCA II occurs in the active‐site of Cyp18, since binding of the inhibitor cyclosporin A abolishes rate acceleration of the refolding reaction. Obviously, the catalytic mechanisms of Cyp18 can differ when acting upon a simple model peptide, four residues long, with easily accessible Pro residues compared with a large protein molecule undergoing folding with partly or completely buried Pro residues. In the latter case, the isomerization kinetics are significantly slower and simpler mechanistic factors such as desolvation and/or strain might operate during folding‐assisted catalysis, since binding to the hydrophobic active site is still a prerequisite for catalysis.     

Proline can have opposite effects on fast and slow protein folding phases

Proline isomerization is well known to cause additional slow phases during protein refolding. We address a new question: does the presence of prolines significantly affect the very fast kinetics that lead to the formation of folding intermediates? We examined both the very slow (10-100 min) and very fast (4 micro s-2.5 ms) folding kinetics of the two-domain enzyme yeast phosphoglycerate kinase by temperature-jump relaxation. Phosphoglycerate kinase contains a conserved cis-proline in position 204, in addition to several trans-prolines. Native cis-prolines have the largest effect on folding kinetics because the unfolded state favors trans isomerization, so we compared the kinetics of a P204H mutant with the wild-type as a proof of principle. The presence of Pro-204 causes an additional slow phase upon refolding from the cold denatured state, as reported in the literature. Contrary to this, the fast folding events are sped up in the presence of the cis-proline, probably by restriction of the conformational space accessible to the molecule. The wild-type and Pro204His mutant would be excellent models for off-lattice simulations probing the effects of conformational restriction on short timescales.     

Proline replacements and the simplification of the complex,parallel channel folding mechanism for the alpha subunit of Trp synthase,a TIM barrel protein

The kinetic folding mechanism for the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli involves four parallel channels whose inter-conversions are controlled by three cis/trans prolyl isomerization reactions (tau(1), tau(2) and tau(3)). A previous mutational analysis of all 19 proline positions, including the unique cis Asp27-Pro28 peptide bond, revealed that the G(3)P28G, P78A or P96A mutations selectively eliminated the fast, tau(1) (ten seconds), folding phase, while the P217M and P261A mutations eliminated the medium, tau(2) (40 seconds) and the slow, tau(3) ( approximately 300 seconds) folding phases, respectively. To further elucidate the role of these proline residues and to simplify the folding mechanism, a series of double and triple mutants were constructed at these critical positions, and comprehensive kinetic and thermodynamic experiments were performed. Although it was not possible to construct a stable system that was free of proline isomerization constraints, a double mutant variant, G(3)P28G/P217M, in which the refolding of more than 90% of the unfolded protein is not limited by proline isomerization reactions was identified. Further, long-range interactions between several of these residues appear to be a crucial part of the cooperative network of structure that stabilizes the TIM barrel motif for alphaTS.